Potentiation of Caspase-1 Activation by the P2X7 Receptor Is Dependent on TLR Signals and Requires NF- B-Driven Protein Synthesis

Department of Pathology, Case School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
The Journal of Immunology (Impact Factor: 4.92). 01/2006; 175(11):7611-22. DOI: 10.4049/jimmunol.175.11.7611
Source: PubMed

ABSTRACT The proinflammatory cytokines IL-1beta and IL-18 are inactive until cleaved by the enzyme caspase-1. Stimulation of the P2X7 receptor (P2X7R), an ATP-gated ion channel, triggers rapid activation of caspase-1. In this study we demonstrate that pretreatment of primary and Bac1 murine macrophages with TLR agonists is required for caspase-1 activation by P2X7R but it is not required for activation of the receptor itself. Caspase-1 activation by nigericin, a K+/H+ ionophore, similarly requires LPS priming. This priming by LPS is dependent on protein synthesis, given that cyclohexamide blocks the ability of LPS to prime macrophages for activation of caspase-1 by the P2X7R. This protein synthesis is likely mediated by NF-kappaB, as pretreatment of cells with the proteasome inhibitor MG132, or the IkappaB kinase inhibitor Bay 11-7085 before LPS stimulation blocks the ability of LPS to potentiate the activation of caspase-1 by the P2X7R. Thus, caspase-1 regulation in macrophages requires inflammatory stimuli that signal through the TLRs to up-regulate gene products required for activation of the caspase-1 processing machinery in response to K+-releasing stimuli such as ATP.

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Available from: George R Dubyak, Nov 02, 2014
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    • "Studies have measured an increase in cytoplasmic ROS upon silica treatment and attributed this to NOX2 activation. The interpretation of this measurement is difficult because cells were preactivated by either phorbol myristate acetate or lipopolysaccharide before exposure to silica, which results in generalized and nonphagosomal NOX activation and an increase in cellular ROS even before exposure to silica (Kahlenberg et al., 2005; Li et al., 2009; Bauernfeind et al., 2011; Brass et al., 2012). Our experiments were therefore performed without preactivating macrophages. "
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    ABSTRACT: Chronic inhalation of silica particles causes lung fibrosis and silicosis. Silica taken up by alveolar macrophages causes phagolysosomal membrane damage and leakage of lysosomal material into the cytoplasm to initiate apoptosis. We have investigated the role of reactive oxygen species (ROS) implicated in causing this membrane damage by studying the spatio-temporal generation of ROS. In macrophages, ROS generated by NADPH oxidase 2 (NOX2) was detected in phagolysosomes containing either silica particles or non-toxic latex particles. ROS was only detected in the cytoplasm of cells treated with silica and appeared in parallel with an increase in phagosomal ROS as well as several hours later associated with mitochondrial production of ROS late in apoptosis. Pharmacological inhibition of NOX activity did not prevent silica induced phagolysosomal leakage, but delayed it. In Cos7 cells that do not express NOX2, ROS was detected in silica containing phagolysosomes that leaked. ROS was not detected in phagolysosomes containing latex particles. Leakage of silica containing phagolysosomes in both cell types was transient and after resealing of the membrane, endolysosomal fusion continued. These results demonstrate that silica particles can generate phagosomal ROS independent of NOX activity and we propose that this silica generated ROS can cause phagolysosomal leakage to initiate apoptosis. © 2015 by The American Society for Cell Biology.
    Molecular Biology of the Cell 09/2015; 26(18). DOI:10.1091/mbc.E15-03-0126 · 4.47 Impact Factor
    • "Inflammasome priming normally occurs downstream of the transcription factor NF-B (Bauernfeind et al., 2009) and essentially involves trans-activation of the various genes that encode the different inflammasome subunits and their cytokine targets. Stimuli known to lead to inflammasome priming include toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS), cytokines such as tumour necrosis factor (TNF), and reactive oxygen species (Bauernfeind et al., 2011; Bauernfeind et al., 2009; Kahlenberg et al., 2005). "
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    ABSTRACT: Inflammasomes are multimeric complexes that facilitate caspase-1-mediated processing of the pro-inflammatory cytokines interleukin(IL)-1β and IL-18. Clinical hypertension is associated with renal inflammation, and elevated circulating levels of IL-1β and IL-18. Therefore, we investigated whether hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and if inhibition of inflammasome activity reduces blood pressure (BP), markers of renal inflammation and fibrosis. Wild-type and inflammasome-deficient ASC(-/-) mice were uninephrectomised and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt). Control mice were uninephrectomised and received placebo and water. BP was measured by tail cuff; renal expression of inflammasome subunits and inflammatory markers was measured by real-time PCR and immunoblotting; macrophage and collagen accumulation was assessed by immunohistochemistry. 1K/DOCA/salt-induced hypertension in mice was associated with increased renal mRNA expression (fold-change vs control; P<0.05) of inflammasome subunits NLRP3 (2.3±0.2), ASC (2.8±0.6) and pro-caspase-1 (2.6±0.5), and the cytokine, pro-IL-1β (4.0±0.8), as well as protein levels of active caspase-1 (1.6±0.2) and mature IL-1β (2.1±0.3). Following treatment with 1K/DOCA/salt, ASC(-/-) mice displayed blunted pressor responses (140±3 vs 155±8 mmHg in wild-types; P<0.05), and were also protected from increases in renal expression of IL-6, IL-17A, CCL2, ICAM-1 and VCAM-1, and accumulation of macrophages and collagen. Finally, treatment with a novel inflammasome inhibitor, MCC950, reversed hypertension in 1K/DOCA/salt-treated mice. Renal inflammation, fibrosis and elevated BP induced by 1K/DOCA/salt-treatment are dependent on inflammasome activity, highlighting the inflammasome/IL-1β pathway as a potential therapeutic target in hypertension. This article is protected by copyright. All rights reserved.
    British Journal of Pharmacology 06/2015; DOI:10.1111/bph.13230 · 4.84 Impact Factor
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    • "A well-characterized route linking TLRs with the NLRP3 inflammasome is via an ATP-gated purinergic receptor, P2X 7 (Mariathasan et al., 2006) which we have previously shown to be expressed and functional in cord blood mononuclear cells (CBMCs) (Warren et al., 2008). While TLR4 leads to increased synthesis of mRNA for the precursor form of IL- 1β, processing to its mature and releasable form is reliant upon the exit of K + from cells—a role performed by the P2X 7 receptor (Kahlenberg et al., 2005). Thus, while numerous studies have shown links between TLR4 and LPS to cytokine release, stimulation with ATP increases IL-1β release further through the P2X 7 -receptor and may be a missing ingredient in understanding sPTB. "
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    ABSTRACT: Distinguishing between fetal and maternal inflammatory responses is necessary for understanding the immune interplay either side of the placenta. Fetal immunity reaches maturity during extrauterine life and while basic inflammatory responses afford a certain degree of protection, fetuses are vulnerable to infection. With the discovery of inflammasomes-intracellular scaffolds that facilitate the elaboration of reactions resulting in the release of mature interleukin-1β (IL-1β)-it is necessary to consider how inflammatory stimuli are processed. The purinergic P2X7 receptor located on haematopoietic cells is a key intermediary in signal transduction initiated at Toll-like receptors (TLR) terminating in release of the mature IL-1β product. We demonstrate herein that IL-1β release from fetal membranes and mononuclear cells isolated from cord, placental, and maternal blood, obtained at term, is P2X7- and caspase-1 dependent. The P2X7-dependent release of the cytokine, which was highest from choriodecidua, was attenuated by progesterone (P4), prolactin and an NFkB inhibitor. The NLRP3 inflammasome appears necessary for the processing of IL-1β in gestational tissues and leukocytes.
    Frontiers in Physiology 06/2015; 6:186. DOI:10.3389/fphys.2015.00186 · 3.53 Impact Factor
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